Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

Abstract

BackgroundThe folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.MethodsPolyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.ResultsAs well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.ConclusionsBoth PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

A novel trafficking pathway in Plasmodium falciparum for the organellar localization of glutathione peroxidase‐like thioredoxin peroxidase

“…2A). This rationale for assigning dual localization of proteins to the apicoplast and mitochondrion has been used in other reports [11,12]. Interestingly, in very few parasites (< 5%), cytosolic localization of PfTPx Gl was also observed in addition to the organellar localization (Fig.…”

“…Two isoforms of serine hydroxymethyltransferase (PfSHMTc and PfSHMTm) in this species show a similar but reportedly stage-specific compartmentalization, with specific isoforms being completely absent in a compartment at a given lifecycle stage [12]. Another such example has been reported in plants with GFP fusions of the sigma factor protein ZmSig2B [30], where the protein was localized in a mutually exclusive manner either to the mitochondria or to the chloroplasts of a specific cell in the ratio 5 : 1.…”

“…This has led us to propose that the ERGolgi route is, in high probability, the route preferred by PfTPx Gl for its two organellar destinations. It would be of interest to assess whether targeting of lipoate protein ligase 2 and the serine hydroxymethyltransferases [11,12] is also via the ER-Golgi route.…”

Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

“…Recently, Read et al [27] reported that the first 100 N-terminal amino acids of Pf mSHMT targets GFP-fusion protein to the mitochondrion. In order to identify the minimal mitochondrion-targeting sequence, plasmids were constructed to express GFP fusion proteins containing a series of truncations of the N-terminal 120 amino acids (Figure 3).…”

“…Previous study observed that the first 100 N-terminus of Pf mSHMT is sufficient for mitochondria targeting [27]. In this study, transfection system using GFP reporter gene was taken to examine the cellular localization of Pf mSHMT and to identify the minimum sequence required for mitochondrial targeting of this enzyme.…”